The cells of eukaryotes, including humans and other mammals, replicate themselves by carrying out an ordered sequence of events, which are cyclically repeated in each successive cell division. In somatic (non germ-line) cells, a typical cycle has four characterized phases: G1, an interval following the completion of mitosis, also termed first gap phase; S, a period during which the cell undergoes DNA synthesis; G2 or second gap phase following completion of DNA synthesis and preceding mitosis; and M, mitosis, where separation of complete sets of replicated DNA occurs. The end result of this process is the generation of two daughter cells that are equivalent both in genetic makeup and in size to the original parent cell. A complex series of biochemical interactions act to control the cell cycle through a series of checkpoints or gating reactions which function to ensure that the requisite precursor phases are completed before the ensuing phase begins. In particular, the checkpoints ensure accurate reproduction and dispersion of the cell's genetic material. In a metazoan organism with differentiated tissues, such as a human being, cells of different tissues replicate at vastly different rates at different life stages. Early embryonic cells replicate rapidly and synchronously, whereas at later stages of development and during adulthood, some cells, such as muscle and nerve cells stop replicating while others, such as epithelial cells, continue to divide throughout the organism's life. Failure of cells to precisely control their replicative state therefore leads to a variety of diseases of pathological proliferation, including cancers and atherosclerosis.
Progress over the past several years has greatly advanced the general understanding of the biochemical reactions which regulate the cell cycle. A general paradigm for cell cycle regulation has emerged in which complexes composed of cyclins and cyclin-dependent kinases (CDKs) regulate progression through stages of the cell cycle. Several mechanisms exist to keep the activity of the cyclin/CDK complexes turned off until the appropriate stage of the cell cycle. Known mechanisms include reversible phosphorylation, binding to small molecular weight inhibitors, transcription control, intracellular location and protein degradation. In yeast, there are multiple cyclins but only a single CDK. The CDK of fission yeast is encoded by the cdc2 gene, that of budding yeast by the cdc28 gene. In higher eukaryotes, including humans, there are multiple CDKs as well as multiple cyclins. Despite the greater complexity of the higher eukaryotes, the overall scheme for cell cycle progression involving cyclins and CDKs is conserved. Deregulation of components of these regulatory pathways has been implicated in human cancer. For a recent general review, see Hunter, T. et al. (1994) Cell 79:573-582.
In humans, there are three known Cdc25 phosphatases, denoted Cdc25A, Cdc25B and Cdc25C. Cdc25B has been shown to be overexpressed in certain breast cancers. In human cells, Cdc25C is present throughout the cell cycle. Its substrate, Cdc2/cyclin B, accumulates throughout the S and G2 phases of the cell cycle. Cdc25C is itself regulated by phosphorylation. The major site of Cdc25C phosphorylation is serine 216 (Ogg, S. et al. (1994) J. Biol. Chem. 269:30461-30469). The protein kinase that acts on Cdc25C was purified over 8000-fold from rat liver. Ogg, S. et al. (1994) J. Biol. Chem. 269:30461.! It was shown to phosphorylate a peptide substrate having the sequence of amino acids 210-231 of human Cdc25C, but not the equivalent peptide in which serine 216 had been changed to a threonine. The kinase is referred to herein as TcAK1, an acronym for twenty-five C associating kinase.
A family of proteins known as 14-3-3 proteins was first identified by Moore, B. F. et al. (1967) as very abundant 27-30 kD acidic proteins of brain tissue (Physiological and Biochemical Aspects of Nervous Integration, F. D. Carlson, Ed., Prentice-Hall, Englewood Cliffs, N.J., 1967). Their name reflects the original investigators' nomenclature. Recent work has implicated the participation of 14-3-3 proteins in cell cycle control (Ford, J. C. et al. (1994) Science 265:533). (For a general review, see Morrison, D. (1994) Science 266:56-57 and Aitken, A. (1995) TIBS 20). A variety of functions have been ascribed to the 14-3-3 proteins. However, several lines of evidence suggest that they link signal transduction cascades with the cell cycle. Various 14-3-3 isoforms have been found in complexes with proteins that transform cells, e.g., the middle T antigen of polyoma virus and Bcr-Ab1, with signaling molecules, e.g., c-Raf1, c-Bcr and P13k, and with two cell cycle regulators, e.g., Cdc25A and CdcB. The ability of a protein to bind or form a complex with a 14-3-3 protein therefore indicates that the protein is a key element linking external or internal signal transduction with other cell functions. In the present invention that other function is the cell replication cycle, specifically passage from G2 to mitosis.